Mechanisms of Virus Entry into Cells and Antiviral Barriers Limiting Entry

自该项目于2017年2月开始工作以来，我们已经生成了大量新的IFITM3突变体来帮助我们的研究。此外，我们开发了基于mlv的载体系统，并采用假分型程序来研究各种逆转录病毒糖蛋白如何影响病毒对ifitm介导的抗病毒活性的易感性。到目前为止，我们的数据表明IFITM3在抑制逆转录病毒颗粒感染性方面表现出很大的广度。这些发现表明，这些抗病毒蛋白可能对不同物种中具有重要医学意义的多种哺乳动物逆转录病毒施加严重的屏障。具体来说，IFITM3在产生病毒的细胞中的存在导致包膜糖蛋白水平降低，这导致病毒颗粒显示很少的糖蛋白，需要与细胞靶标结合并进行融合。这项工作的第一部分已经发表（Ahi et al., mBio 11: e03088- 19,2020）。我们证明了IFITM3在外源性或内源性表达时对病毒糖蛋白具有负调控作用，因为RNAi和CRISPR/ cas9介导的基因敲除人细胞和小鼠细胞中的IFITM3会导致病毒糖蛋白的半衰期延长。此外，IFITM3通过干扰病毒糖蛋白的顺行运输并将其重定向到溶酶体进行降解来抑制病毒糖蛋白。我们发现病毒糖蛋白的抑制是广泛的（影响HIV-1 Env， MLV Env和vsv -糖蛋白），而埃博拉病毒糖蛋白具有耐药性。此外，许多细胞糖蛋白不受IFITM3的影响。这一发现表明IFITM3利用一种通用机制广泛抑制病毒融合和传播所需的病毒糖蛋白的产生。我们还发现了一些失去这种抗病毒功能的IFITM3突变体。我们目前正在使用结构同源性来预测这些突变位点所属的功能基序，这将有助于我们理解单个突变如何以及为什么破坏活性。最近，我们在细胞内环（G95L）中发现了一个单独的残基突变，该突变取消了这种抗病毒功能。我们发现G95形成了蛋白质寡聚化所需的二甘氨酸基序的一部分。我们的研究结果表明，IFITM3寡聚物抑制了其抗病毒活性（抑制病毒进入初始细胞和抑制病毒粒子在病毒产生细胞中的感染性）（Rahman等人，提交）。总之，这些努力将为IFITM蛋白及其所在的CD225蛋白家族的功能提供广泛的见解，并将为开发新的抗病毒疗法提供杠杆作用。

Since work on this project begun in February 2017, we have generated a large panel of novel IFITM3 mutants to aid our study. Furthermore, we have developed systems using MLV-based vectors and adapted a pseudotyping procedure to study how various retroviral glycoproteins affect viral susceptibility to IFITM-mediated antiviral activities. Our data thus far suggest that IFITM3 exhibits great breadth with regard to its ability to inhibit retroviral particle infectivity. These findings suggest that these antiviral proteins may impose serious barriers to multiple mammalian retroviruses of medical importance in different species. Specifically, the presence of IFITM3 in virus-producing cells leads to decreased levels of envelope glycoprotein, which results in virus particles displaying very little glycoprotein needed to attach and perform fusion with cell targets. The first installment of this work has been published (Ahi et al., mBio 11: e03088-19, 2020). We demonstrated that IFITM3 negatively regulates viral glycoproteins when expressed ectopically or endogenously, as RNAi and CRISPR/Cas9-mediated gene knockout of IFITM3 in human cells and in murine cells results in longer half-lives of viral glycoproteins. Furthermore, IFITM3 inhibits viral glycoproteins by interfering with their anterograde trafficking and redirecting them to lysosomes for degradation. We found that inhibition of viral glycoproteins is broad (affecting HIV-1 Env, MLV Env, and VSV-glycoprotein), while Ebolavirus glycoprotein is resistant. Furthermore, a number of cellular glycoproteins are unaffected by IFITM3. This finding reveals that IFITM3 utilizes a general mechanism to broadly inhibit the production of viral glycoproteins needed for virus fusion and spread. We have also identified a number of IFITM3 mutants that have lost this antiviral function. We are currently using structural homology to predict functional motifs to which these mutated sites belong, which will facilitate our understanding of how and why individual mutations disrupt activity. Most recently, we identified a single residue mutation in the intracellular loop (G95L) that abrogates this antiviral function. We found that G95 forms part of a di-glycine motif needed for protein oligomerization. Our findings indicate that IFITM3 oligomers are responsible for the inhibition of both of its antiviral activities (inhibition of virus entry into naive cells and inhibition of virion infectivity in virus-producing cells) (Rahman et al., submitted). Together, these efforts will provide extensive insight into the function of IFITM proteins as well as the extended CD225 protein family to which they belong and will provide leverage for the development of new antiviral therapies.